Analyses of Volatiles Produced by the African Fruit Fly Species Complex (Diptera, Tephritidae)
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A peer-reviewed open-access journal ZooKeysAnalyses 540: 385–404 of volatiles (2015) produced by the African fruit fly species complex( Diptera, Tephritidae) 385 doi: 10.3897/zookeys.540.9630 RESEARCH ARTICLE http://zookeys.pensoft.net Launched to accelerate biodiversity research Analyses of volatiles produced by the African fruit fly species complex (Diptera, Tephritidae) Radka Břízová1,2, Lucie Vaníčková1,3, Mária Faťarová4, Sunday Ekesi5, Michal Hoskovec1, Blanka Kalinová1,6 1 Institute of Organic Chemistry and Biochemistry ASCR, v.v.i., Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic 2 Institute of Chemical Technology in Prague, Technická 5, CZ-166 28 Prague 6, Czech Re- public 3 Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Av. Lourival de Melo Mota, s/n, Tabuleiro, CEP 57072-970, Maceió, AL, Brazil 4 Charles University, Albertov 6, CZ-128 43 Prague 2, Czech Republic 5 International Centre of Insect Physiology and Ecology, PO Box 30772-00100 GPO, Nairobi, Kenya 6 Czech University of Life Sciences, Kamýcká 129, CZ-165 21 Prague 6, Czech Republic Corresponding author: Lucie Vaníčková ([email protected]) Academic editor: M. De Meyer | Received 20 March 2015 | Accepted 10 June 2015 | Published 26 November 2015 http://zoobank.org/2A59E102-E36C-4A5D-94F6-4056A6EACED4 Citation: Břízová R, Vaníčková L, Faťarová M, Ekesi S, Hoskovec M, Kalinová B (2015) Analyses of volatiles produced by the African fruit fly species complex (Diptera, Tephritidae). In: De Meyer M, Clarke AR, Vera MT, Hendrichs J (Eds) Resolution of Cryptic Species Complexes of Tephritid Pests to Enhance SIT Application and Facilitate International Trade. ZooKeys 540: 385–404. doi: 10.3897/zookeys.540.9630 Abstract Ceratitis fasciventris, Ceratitis anonae and Ceratitis rosa are polyphagous agricultural pests originating from the African continent. The taxonomy of this group (the so-calledCeratitis FAR complex) is unclear. To clarify the taxonomic relationships, male and female-produced volatiles presumably involved in pre- mating communication were studied using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) followed by multivariate analysis, and gas chro- matography combined with electroantennographic detection (GC-EAD). GC×GC-TOFMS analyses re- vealed sex specific differences in produced volatiles. Male volatiles are complex mixtures that differ both qualitatively and quantitatively but share some common compounds. GC-EAD analyses of male volatiles revealed that the antennal sensitivities of females significantly differ in the studied species. No female volatiles elicited antennal responses in males. The results show clear species-specific differences in volatile production and provide complementary information for the distinct delimitation of the putative species by chemotaxonomic markers. Copyright Radka Břízová et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 386 Radka Břízová et al. / ZooKeys 540: 385–404 (2015) Keywords Ceratitis FAR complex, chemotaxonomy, male and female-borne volatiles, GC×GC-TOFMS, GC-EAD Introduction The fruit fly family Tephritidae (Diptera) consists of four major genera,Ceratitis , Bac- trocera, Anastrepha and Rhagoletis, which are considered important insect pests world- wide (Aluja and Norrbom 2001). The genusCeratitis is the most studied, with Ceratitis capitata (Wiedemann) as species most frequently monitored and used as the model pest organism due to its global distribution. The Afro-tropical group of fruit flies Ceratitis fasciventris (Bezzi), C. anonae Graham and C. rosa Karsch (the so-called FAR complex) is widespread in a number of African countries: C. fasciventris and C. anonae occur sym- patrically in both East and West Africa while C. rosa is more restricted to southern and eastern Africa where its distribution partially overlaps with that of C. fasciventris but not with C. anonae (Barr and McPheron 2006, Copeland et al. 2006). Ceratitis rosa is now feared to be a global threat due to its tolerance to lower temperatures (Duyck and Quilici 2002). It may expand not only within Africa, but also across Europe, Asia, Australia and the North and South American continents (De Meyer et al. 2008). Females of the FAR complex species cause extensive damage on commercially pro- duced fruits from 24 plant families (De Meyer et al. 2002, Copeland et al. 2006), by puncturing the fruits during oviposition and by the feeding larvae inside the fruit that generally result in premature fruit abortion. Reproduction behavior in genus Ceratitis is initiated by males that aggregate in leks on vegetation to lure females by releasing long-range pheromones. Females attracted by male pheromone visit leks and choose males to mate based on complex visual, acoustical and chemical stimuli (Aluja and Norrbom 2001). Chemical communication, that involves both long-range phero- mones and close-range cuticular hydrocarbons, is integral part of the fruit fly court- ship. Nevertheless, there are no records available on the composition of the long-range volatiles released by the FAR complex species. Despite its economic importance, the taxonomy of this group is not clear and taxonomical classification is not easy (De Meyer and Freidberg 2006). It is particularly important to resolve invasive agricultural pest species, because inadequate morpho- logical/molecular characterisation of the species might have serious economic conse- quences, resulting in inept ecological models and/or pest control strategies (Virgilio et al. 2013, Vaníčková et al. 2014). The need to develop a precise pest-detection technique, diagnostic tools and man- agement strategies for these pest species initiated large scale morphological and genetic studies, the investigation of their evolutionary relationships as well as the characterisa- tion of the variation of cuticular hydrocarbon profiles within and between the species (De Meyer 2001, De Meyer and Freidberg 2006, Virgilio et al. 2012, Delatte et al. 2013, Virgilio et al. 2013, Vaníčková et al. 2014a,b, Vaníčková et al. 2015). The species of the FAR complex can only be identified based on specific small differences between Analyses of volatiles produced by the African fruit fly species complex( Diptera, Tephritidae) 387 the morphological characters of adult male leg patterns (larvae, pupae and females, are even more difficult to distinguish) (De Meyer and Freidberg 2006). While it is possible to identify the females of C. anonae, the females of C. rosa and C. fasciventris are almost indistinguishable from each other and show only very subtle differences in their scutel- lar colour patterns. The absence of clear diagnostic morphological features to identify individual species emphasises the need for unambiguous identification applying molec- ular and/or chemical tools (Baliraine et al. 2004, Vaníčková 2012, Virgilio et al. 2013, Vaníčková et al. 2014a,b). Molecular approaches for species recognition were developed in the past (Virgilio et al. 2008, Barr and Wiegmann 2009, Delatte et al. 2013). Recent- ly, Virgilio and co-workers have provided clear data on the specification of five different morphotypes using a comparison of allelic variations at 16 microsatellite loci (Virgilio et al. 2013). Nevertheless, the use of microsatellite loci for cryptic species identification is rather laborious and expensive. Recent studies on the cuticular hydrocarbon profiles extracted from the body surface of males and females of C. fasciventris, C. anonae, C. rosa and C. capitata have supported the existence of more than three genotypes in the FAR complex (Vaníčková 2012, Vaníčková et al. 2014a,b, Vaníčková et al. 2015). The authors pinpointed several chemotaxonomic markers whose presence/absence can be used for the identification of the putative species from the FAR complex. To understand the detailed taxonomical relationships within the FAR complex and to support the evidence on cryptic speciation presented in the aforementioned studies, we aimed to analyse the chemical composition of the volatiles emitted by males and females. The communication signals are highly species-specific and are ex- tremely important in the reproduction isolation of different species. Therefore we as- sume that the specific volatiles, examined in the present study, together with cuticular hydrocarbons could serve as an effective diagnostic tool. Methods Insects The laboratory-reared populations of Ceratitis fasciventris, C. anonae and C. rosa R2 type were obtained from the International Centre of Insect Physiology and Ecology (ICIPE, Nairobi, Kenya). The pupae were kept under identical laboratory conditions at the In- stitute of Organic Chemistry and Biochemistry (IOCB, Prague, Czech Republic). Adult flies were fed on an artificial diet consisting of sugarcane:yeast (3:1) and mineral water and were kept at a relative humidity of 60%, at 25 °C, and a 12L:12D photoperiod. The collection of volatiles Male and female-borne volatiles of all three species were trapped by the standard dy- namic headspace procedures. A group of five virgin 20-day-old male and/or female flies 388 Radka Břízová et al. / ZooKeys 540: 385–404 (2015) of each species was placed into round-bottom flasks (250 mL) adapted for volatile col- lection (Verkon, Praha, Czech Republic). Air was sucked by a pump (Pocket